Project Summary Currently, about 500 clinically distinct skeletal dysplasias have been described, and the genetic basis of 2/3 of them has been revealed. However, patients with genetic bone diseases, such as osteogenesis imperfecta (OI), have limited therapeutic options despite the fact that its genetics and pathophysiology have been well described. For genetic bone diseases, replacing mutated bone cells by healthy cells is one of most straightforward therapeutic strategies. Cell-based therapy was tired to several genetic bone diseases including OI, and it was shown that transplantation of bone marrow (BM) cells or BM-derived mesenchymal stromal cells (MSCs) ameliorated the condition of patients and mice with OI. However, cell transplantation therapy has not been actively pursued for multiple reasons, including poor donor-cell contribution to bone after systemic administration in clinical and pre-clinical studies. For cell-based therapy to be safe and effective, achieving efficient long-term engraftment and use of patient-derived cells appear important. To achieve this goal, we propose to utilize a rare bone stromal population that includes long-term osteoprogenitor cells in combination with an efficient allele-specific genome-editing system. This idea is based on the findings that a rare Sox9-positive cell population contains long-term osteoprogenitor cells that give rise to MSCs. Since these cells self-renewing progenitors in vivo, they likely show long-term engraftment. In addition, their small size will make them more suitable than large MSCs for systemic administration. However, since these cells are rate, it is necessary to amplify them for further application. In R61, we will develop methods to amplify Sox9-positive cells by ex vivo expansion by optimizing the condition that stimulates asymmetric cell division and by reprogramming from MSCs using non-genetic methods. We then evaluate their transplantability to bone. To use autografts for treatment, the mutation of patient-derived Sox9-positive cells needs to be repaired. In R33, to demonstrate the proof-of-concept, we will use Aga2 OI model mice that have a heterozyous point mutation in the type I collagen gene. First, we will develop an efficient, mutation-specific genome editing method by combining the new CRISPR tool, adenosine base editor (ABE) and the high capacity adenovirus gene transfer system to repair the Aga2 mutation in Sox9-postive cells from mutant mice. We will then evaluate the therapeutic effect of transplantation with genome-edited Aga2 Sox9-postive cells in Aga2 mice. This project will not only translational impacts by changing the cell-based therapy for bone diseases but also provide unique research materials to investigate osteoprogenitor biology. !